On-chip hybridization coupled with ITP based purification for fast sequence specific identification

Abstract

Isotachophoresis (ITP) can be employed to simultaneously focus the target and ligand of an assay into the same ITP focus zone. The target and ligand can bind to each other in the ITP focus zone, and then the resulting bound complex can be detected (e.g., by fluorescence). The sensitivity of this approach can be greatly increased by the enhanced concentration of both target and ligand that ITP provides in the focus zone. Since ITP can be performed quickly, the resulting assay is both rapid and sensitive. Markers of bacterial urinary tract infections have been experimentally detected at clinically relevant concentrations with this approach. MicroRNA sequences have also been profiled with this approach, which is clinically relevant because MicroRNA is expected to provide useful markers for disease. In one experiment, miR- 122 in human kidney and liver was detected and quantified.

The invention claimed is: 
     
       1. A method for sample analysis and/or preparation, the method comprising:
 performing isotachophoresis (ITP) on a sample including at least one target species, wherein the target species is localized to a first ITP focus zone by the isotachophoresis; 
 providing at least one ligand to the isotachophoresis such that the ligand is localized to the first ITP focus zone by the isotachophoresis, wherein the target species and ligand bind to each other in the first ITP focus zone to form a bound complex; 
 wherein the ligand and/or the target comprises a nucleotide sequence; and 
 performing analysis of the bound complex to provide information on the target species. 
 
     
     
       2. The method of  claim 1 , wherein the ligand comprises at least one molecular beacon molecule having a fluorescence signal that increases substantially upon hybridization. 
     
     
       3. The method of  claim 2 , wherein the molecular beacon has a first end comprising a fluorophore and a second end comprising a quencher, wherein the first end is in sufficient proximity to the second end when the molecular beacon is not hybridized for the quencher to substantially suppress fluorescence from the fluorophore, and wherein the first end is sufficiently far from the second end when the molecular beacon is hybridized for fluorescence from the fluorophore to be substantially unaffected by the quencher. 
     
     
       4. The method of  claim 1 , wherein an effective mobility of the ligand and an effective mobility of the bound complex differ. 
     
     
       5. The method of  claim 4 , wherein the bound complex remains in the first ITP focus zone. 
     
     
       6. The method of  claim 4 , wherein the bound complex focuses in a second ITP focus zone distinct from the first ITP focus zone. 
     
     
       7. The method of  claim 4 , wherein the bound complex is not focused by the isotachophoresis. 
     
     
       8. The method of  claim 4 , further comprising separating the ligand, the target and/or the bound complex after formation of the bound complex. 
     
     
       9. The method of  claim 8 , wherein the separating the ligand, the target and/or the bound complex after formation of the bound complex comprises one or more methods selected from the group consisting of: isotachophoresis, electrophoresis, and chromatography. 
     
     
       10. The method of  claim 1 , wherein the at least one ligand provides a cooperative labeling of target species that bind two or more ligand molecules. 
     
     
       11. The method of  claim 10 , wherein the cooperative labeling is based on Förster resonance energy transfer (FRET), wherein a first bound probe molecule and a second bound probe molecule are adjacent to each other along the target species within the bound complex, wherein the first bound probe molecule includes a first fluorophore in proximity to the second bound probe molecule, wherein the second bound probe molecule includes a second fluorophore in proximity to the first bound probe molecule, and wherein excitation of the first fluorophore leads to emission from the second fluorophore by FRET. 
     
     
       12. The method of  claim 1 , wherein the sample further includes at least one precursor species of the at least one target species, and wherein the precursor species is not focused in the first ITP focus zone of the target species. 
     
     
       13. The method of  claim 12 , wherein the precursor species is also capable of binding to the ligands, whereby signals from the target species and the precursor species are spatially separated. 
     
     
       14. The method of  claim 1 , wherein the ligand is labeled with a fluorescent label. 
     
     
       15. The method of  claim 1 , wherein the target species is labeled with a fluorescent label, and wherein the ligand includes a quencher for the fluorescent label. 
     
     
       16. The method of  claim 1 , wherein the ligand comprises at least one nucleotide hybridization probe which is fluorescently labeled. 
     
     
       17. The method of  claim 16 , wherein the at least one target species is selected from the group consisting of: nucleic acid species, peptides capable of binding to a nucleic acid, polypeptides capable of binding to a nucleic acid, and proteins capable of binding to a nucleic acid. 
     
     
       18. The method of  claim 16 , wherein the at least one nucleotide hybridization probe is selected from the group consisting of: nucleic acid probes and aptamers. 
     
     
       19. The method of  claim 1 , further comprising partially hybridizing the target species and ligand prior to performing the isotachophoresis. 
     
     
       20. The method of  claim 1 , further comprising extracting bound complex from the first ITP focus zone. 
     
     
       21. The method of  claim 1 , wherein the analysis of the bound complex is performed in the first ITP focus zone. 
     
     
       22. A method for clinically screening for disease, the method comprising:
 obtaining a patient specimen; 
 performing the method of  claim 1 , wherein the sample is derived from the patient specimen, and wherein the ligand is capable of binding to a target species that is a marker for disease.